Three-dimensional orientation determination of single molecules by emission pattern characterization

Abstract

Summary form only given. We have employed wide-field total internal reflection (TIR) through a high numerical aperture microscope objective to simultaneously excite many individual x, y, and z-oriented fluorescent DiIC/sub 18/ molecules. In an ideal imaging system, all fluorophores appear as identical, diffraction-limited Airy patterns. Deliberate optical departures from ideality induce slight aberrations into the optical path, recovering normally obscured 3-D orientational information through direct imaging of single molecule dipole emission patterns. Because molecules emit with a characteristic sine-squared emission pattern with respect to the transition dipole direction, observation of these fluorescent patterns is indicative of both single molecule behavior and true 3-D molecular orientation.